1. Field of the Invention (Technical Field)
The present invention relates to non-cellular display of 7-transmembrane receptors on beads or in suspension, and their use in flow cytometry or multi-well fluorescence or resonance energy transfer to evaluate ligand discovery, especially within combinatorial libraries. The invention is also a method to use the above constructs and protocols to detect real-time receptor-G-protein interactions or interactions between receptors and other intracellular components.
2. Background Art
Much of modern biomedical research, including drug discovery, involves the analysis of molecular interactions, such as those between receptors and ligands, enzymes and substrates, and drug compounds and their cellular targets. Receptors are of particular interest, as signal transduction via these biological mediators controls such processes as cell growth, movement and function. Development of a system for homogeneous receptor study would allow analysis of stoichiometry, affinity, and kinetics, as well as the elucidation and characterization of signal transduction complexes.
One of the largest families of receptors in the human genome is that of the 7 transmembrane (7 TMR) superfamily, also known as G-protein coupled receptors, numbering approximately 2000. More than 40% of the current drugs on the market target one or more of these receptors. One of the better studied of these receptors is the N-formyl peptide chemoattractant receptor (FRP) and it serves a model system for the entire family. It is largely responsible for numerous immune functions. In addition, 7-TMR have been shown to be docking sites for HIV entry into white blood cells, and are known to be important in asthma as well as the diagnosis and treatment of neuro-endocrine cancer.
7-TMR 10 have seven transmembrane α-helical domains 12, with three connecting loops on each inner and outer face of membrane 14, as shown in FIG. 1. The N-terminal region is extracellular, while the C-terminus is intracellular. The three extracellular loops and transmembrane region participate in ligand binding. Ligands that can stimulate (agonistic) or inhibit (antagonistic) receptor function are primary targets in drug discovery. The intracellular loops, especially the second intracellular loop, and tail, in contrast, participate in interactions with the G-protein. G-proteins are important effectors of cell activation, for example, through the interaction with formyl-peptide receptor-ligand complexes. The pathway of cell activation for monovalent chemoattractant ligands appears to involve the interaction of receptor-ligand complexes with guanine nucleotide-binding proteins (G-proteins). For example, the formyl peptide receptors and other 7TMR in permeabilized cells or cell membranes are sensitive to guanine nucleotides and are able to couple with G-protein. Sklar et al., Regulation of Ligand-Receptor Dynamics for Guanine Nucleotides, 262 J. of Biol. Chem. 135–139 (1987).
Traditional methods for examining receptor behavior require a separation step, frequently involving centrifugation or filtration. These steps are not optimal for real-time kinetic analysis of rapidly equilibrating systems.
Earlier assays were developed to study binding interactions. These include U.S. Pat. No. 4,275,149, to Litman et al., entitled Macromolecular Environment Control in Specific Receptor Assays, which discloses the use of beads, and enhancement or diminution of signal (i.e. diffusion or pH change) due to a receptor-ligand interaction, through the use of chromagen and anti-chromagen molecules. The assay does not allow quantitation or elucidation of actual binding events.
U.S. Pat. No. 4,665,020, to Saunders, entitled Flow Cytometer Measurement of Binding Assays, discloses receptors bound to large beads and ligands bound to smaller beads with a label. The two sizes of beads are added together, and analyzed by flow cytometry for largest size of aggregates, representing bound receptor/ligand complexes. This assay eliminates the need for a washing step, but does not have the ability to assess a library of ligands simultaneously bound to beads.
U.S. Pat. No. 5,747,349, to van den Engh et al., entitled Fluorescent Reporter Beads for Fluid Analysis, discloses reporter molecules bound to a fluorescent bead which is sensitive to some aspect of the analyte e.g., pH or oxygen saturation, causing a change in fluorescence. This assay does not detect aggregates.
U.S. Pat. No. 5,405,784, to Van Hoegaerden, entitled Agglutination Method for the Determination of Multiple Ligands, discloses the use of antiligands on latex beads to analyze substances. Different ligands are associated with fluorescence of different colors. This assay does not allow for bound or free receptors to identify ligands on libraries.
U.S. Pat. No. 5,601,992, to Lerner et al., entitled Peptide Library Formats and Methods Relating Thereto, and U.S. Pat. No. 5,698,685, to Summerton et al., entitled Morpholino-Subunit Combinatorial Library and Method, also do not entail a method to quantitate and elucidate specific receptors, and cannot be used with flow analysis for real-time kinetic analysis.
The above inventions lack the ability to detect ligands and drug interactions in real-time kinetic assays, and do not examine the possibilities of such assays with 7-TMR. The present invention successfully addresses these issues by utilizing beads or micelles to display 7-TMR for flow cytometry and resonance energy transfer (RET) assays to determine the effect various drugs (expressed in combinational libraries or individually in solution) have on the binding capacity, and ultimately the enzymatic activities in receptor signal transduction and termination. It also allows for examination of molecular mechanisms with purified proteins under physiologically meaningful conditions and with known stoichiometry. The display and assays provided by the present invention allow an important sequence of signaling events (ligand binding, receptor and G-protein coupling, and receptor desensitization) to be evaluated as drug targets.